Morphological and molecular characterization of the rust fungus, Phakopsora apoda causing leaf rust on Brachiaria grass in Rwanda

Abstract

Brachiaria grass is one of the major forages that plays a key role in push pull technology. Different diseases including leaf rust caused by rust fungus, Phakopsora apoda have recently become an important challenge resulting in yield loss of Brachiaria grass in different countries including Rwanda. The objective of this study was to provide morphological and molecular characteristics of Phakopsora apoda causing Brachiaria leaf rust. Spores isolated from rusted Brachiaria leaves collected in five districts of Rwanda were analyzed. Samples were put in paper bags and left at room temperature for two days. Thereafter, spores were removed using brushes and put in eppendorf tubes, stored at 4oC in darkness for further characterization. For morphological characteristics, colour and measurements of spores were recorded using Optika B-350 microscope with installed camera and calibrated micrometer. Multigene analysis using rust specific primers and large subunit (LSU) of nuclear ribosomal RNA was performed for molecular characterization. The results indicated that the colour of spores was yellowish or brownish while the shape was ellipsoidal or circular with average measurements of 25.1μm and 16.8μm for length and width respectively. Primers amplified the size of DNA between 1291bp and 1381bp and the size between 874bp to 882bp for rust specific primers and LSU respectively. The identity of the sequence was 96% with e-value of 0, sequence coverage of 60% for rust primer and the sequence identity was 94.97% to 95.37%, sequence coverage of 99% to 100% and e-value of 0 for LSU primer. All sequences recovered from five isolates of leaf rust matched to the genebank accession number MG461668.1 for both rust specific and LSU sequences. The results of this study provide useful information to develop effective options for leaf rust disease management in Rwanda.

Key words: Internal transcribed spacer, large subunit, nuclear ribosomal RNA, Phakopsora apoda, sequence, spore.